Suppression of CINNAMOYL- CoA REDUCTASE increases the level of monolignol ferulates incorporated into maize lignins

Abstract

Background: The cell wall polymer lignin provides structural support and rigidity to plant cell walls, and therefore to the plant body. However, the recalcitrance associated with lignin impedes the extraction of polysaccharides from the cell wall to make plant-based biofuels and biomaterials. The cell wall digestibility can be improved by introducing labile ester bonds into the lignin backbone that can be easily broken under mild base treatment at room temperature. The FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme, which may be naturally found in many plants, uses feruloyl-CoA and monolignols to synthesize the ester-linked monolignol ferulate conjugates. A mutation in the first lignin-specific biosynthetic enzyme, CINNAMOYL-CoA REDUCTASE (CCR), results in an increase in the intracellular pool of feruloyl-CoA.

Results: Maize (Zea mays) has a native putative FMT enzyme, and its ccr mutants produce an increased pool of feruloyl-CoA that can be used for conversion to monolignol ferulate conjugates. The decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue.

Conclusions: Together, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops.

Keywords: Biofuels; Cell wall digestibility; Mass spectrometry; Zea mays.

Fig. 1

An abbreviated version of the lignin biosynthetic pathway highlighting the effects of the ccr1 mutation. The mutated CCR1 gene results in reduced CCR1 transcript, and presumably less CCR1 enzyme, and a pool of feruloyl-CoA (FA reservoir). The conjugates formed between the monolignols and feruloyl-CoA by ZmFMT are incorporated into the lignin polymer, generating a zip-lignin in which the ester bonds in the backbone are readily cleaved by base

Fig. 2

Biomass and lignin monomer composition data from wild-type and Zmccr1 mutant plants. a The Zmccr1 mutant plants appear no different from wild type in growth and biomass yield when grown in the greenhouse. b The levels of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monomers released by thioacidolysis are all significantly lower in the Zmccr1 mutants relative to those in wild-type (WT) plants. Error bars represent standard deviations of biological replicates (n = 5 plants), asterisks represent a significant difference from WT, p < 0.05

Fig. 3

The levels of monolignol conjugates released from DFRC analysis. a The level of monolignol dihydroferulate (ML-DHFA) conjugates released from Zmccr1 mutants is three to fivefold higher than from wild-type (WT) plants, indicating higher ML-DHFA production and incorporation into the lignin polymer. b The level of monolignol dihydro-p-coumarates (ML-DHpCA) is similar between the Zmccr1 mutants and the wild-type plants, indicating that the production of monolignol ferulates and p-coumarates are independent. CA-FA/pCA is the coniferyl alcohol conjugate, SA-FA/pCA the sinapyl alcohol conjugate. Error bars represent standard deviation of biological replicates (n = 5 plants for greenhouse lines, n = 2 pools of 10 plants for field-grown lines), asterisks represent a significant difference from WT, p < 0.05

Fig. 4

Relative glucose and pentose release from limited-extent digestibility of Zmccr1 mutant lines is higher than from wild-type (WT) plants, both in greenhouse and field-grown maize. Error bars represent standard deviations of biological replicates (n = 5 plants for greenhouse lines, n = 2 pools of 10 plants for field-grown lines), asterisks represent significant difference from WT, p < 0.05